Citação Artigo 2 Flavonoides e Carotenoides IFRJ - 2 PDF

Chapter 16
Current Trends in Pteridophyte Extracts:

From Plant to Nanoparticles
Liliana Cristina Soare and Nicoleta Anca Şuţan
16.1 Introduction
The diversity of pteridophytes is estimated at 10,000–15,000 species (Akeroyd and

Synge 1992; Roos 1996), with a distribution of species that increases from the Poles
towards the equator (Moran 2004). Areas of high diversity are found in the Andes
(with approximately 2500 species of ferns and lycophytes), New Guinea (approxi-
mately 2000 species), Borneo (1200 species), Ecuador (1250 species), Costa Rica
(1165 species) (Moran 2008), China (2600 species) (Lu 2007), the Philippines
(1000 species), Malaysia (550 species), Thailand (700 species), India (600 species)
(Lu 2007; Schneider et al. 2004), etc.
Such areas of high pteridophyte biodiversity stimulate the use of a great number
of species for human consumption or in traditional medicine. Thus, in countries
such as Malaysia, the Philippines, Hawaii, Australia, Nepal, India etc., ferns are
used for consumption (Liu et al. 2012). China is home to “more than 20% of the
global pteridophyte flora” (Lu 2007). For over three millennia, 52 edible fern spe-
cies have been used for consumption in China, and other 144 species may be used
for this purpose. Their dry and salted leaves, the starch or the tea made from these
leaves are commercial products in China (Liu et al. 2012).
The use of pteridophytes in the multimillennial traditional medicine of many
people has been the foundation of modern therapeutical research. Crude extracts,
standardized extracts or purified substances have been tested to discover new prop-
erties with therapeutic potential.
The current research methods, techniques and equipment allow us to obtain
results which help validate ethnopharmacological treatments. The identification of
secondary fern metabolites, specific to this group, explains their bioactivity. Modern
research studies have shown that Blechnum orientale (the Centipede fern), an edible
L. C. Soare (*) · N. A. Șuțan

Department of Natural Sciences, University of Piteşti, Pitești, Romania
© Springer International Publishing AG, part of Springer Nature 2018 329

H. Fernández (ed.), Current Advances in Fern Research,
https://doi.org/10.1007/978-3-319-75103-0_16
330 L. C. Soare and N. A. Șuțan
species used in the Asian traditional medicine for various skin diseases, stomach
pain, gall bladder disorders, etc., may be used in the treatment of colon cancer and
that of multidrug-resistant bacterial infections (methicillin resistant Staphylococcus
aureus, MRSA) (Lai et al. 2010) as well as in the treatment of diabetic ulcer wounds
(Lai et al. 2016).
New technologies for identifying pteridophyte species of medicinal importance
have been discovered and developed. Thus, the classical identification of medicinal
fern species based on their morphological characters has been replaced by “DNA
barcoding” (Ma et al. 2010).
Some of the current problems identified in the medical field may be solved by
using natural products obtained from ferns, products that possess a wide range of
biological properties (antibacterial, antifungal, antiviral, antioxidant, anti-
inflammatory, antitumor, hypoglycemic, neuroprotective, etc.). One of the problems
that poses a major threat to the population is the rapid increase in the resistance of
microorganisms to synthetic antibiotics, including those that were recently discov-
ered. Superbacteria, some of them resistant to more than 20 antibiotics, have
appeared in recent years and added to the range of multidrug-resistant bacteria. In
2013, The Centers for Disease Control and Prevention (CDC; https://www.cdc.
gov/) published a study on the “Current Antibiotic Resistance Threats in the United
States, by microorganism”, which mentions three groups of microorganisms:
1. Microorganisms with a threat level of urgent (Clostridium difficile, Carbapenem-
resistant Enterobacteriaceae, drug-resistant Neisseria gonorrhoeae)
2. Microorganisms with a threat level of serious (multidrug-resistant Acinetobacter,
drug-resistant Campylobacter, fluconazole-resistant Candida, extended spec-
trum β-lactamase producing Enterobacteriaceae (ESBLs), vancomycin-resistant
Enterococcus VRE, multidrug-resistant Pseudomonas aeruginosa, drug-resistant
non-typhoidal Salmonella, drug-resistant Salmonella typhi, drug-resistant
Shigella, methicillin-resistant Staphylococcus aureus MRSA, drug-resistant
Streptococcus pneumoniae, drug-resistant tuberculosis)
3. Microorganisms with a threat level of concerning (vancomycin-resistant

Staphylococcus aureus VRSA, erythromycin-resistant Group A Streptococcus,
clindamycin-resistant Group B Streptococcus)
16.2 Crude Extracts
The impressive number of research studies on fern use in traditional medicine has
inspired researchers to create databases that provide information on “medicinal
uses, chemical constituents as well as protein/enzyme sequences” of some species
(Thakar et al. 2015). The information stored in such databases can help us not only
to discover new medicinal drugs but also to conserve pteridophytes (Thakar et al.
2015), in case the species used for therapeutic purposes are rare, vulnerable, etc. and
they would consequently require protection.
16 Current Trends in Pteridophyte Extracts: From Plant to Nanoparticles 331
Extracts were obtained, and phytochemical characterization along with bioactiv-

ity testing was performed for different spontaneously occurring species, such as
Stenochlaena palustris (swampy land, Kampar, Malaysia) (Chai et al. 2012), for
species cultivated in botanical gardens, for example, Blechnum orientale (Putrajaya
Botanical Garden, Kuala Lumpur, Malaezia) (Lai et al. 2010), Azolla microphylla
(Centre for Conservation and Utilization of Blue Green Algae, Indian Agricultural
Research Institute, New Delhi) (Abraham and Kaushik 2015), Psilotum nudum,
Nephrolepis biserrata and N. cordifolia (fern house of National Botanical Institute,
Lucknow, India) (Rani et al. 2010), or for in vitro plants, for example, the gameto-
phyte of Asplenium trichomanes, A. trichomanes-ramosum and Polypodium vulgare
(Deliu et al. 2013), the gametophyte and sporophyte of P. vulgare or the gameto-
phyte of Cystopteris fragilis (Soare et al. 2012a).
Plant parts (leaves, rhizomes, roots, spores or gametophytes) or the whole plant
were used to obtain extracts. The extracts from the whole plant of Dicranopteris
linearis (Rajesh et al. 2016) were tested to establish their phytochemical composi-
tion and antioxidant activity. Cao et al. (2013) used the whole plant of Dryopteris
erythrosora to obtain extracts, which were tested for their antioxidant, anticarcino-
genic and acetylcholinesterase inhibitory properties. The analysis also comprised
the characterization of the flavonoids encountered in these extracts. The leaves of
Blechnum orientale (Lai et al. 2010), Asplenium adiantum-nigrum and A. tricho-
manes (Hammami et al. 2016), A. nidus (Nath et al. 2013), Acrostichum aureum,
Asplenium nidus, Blechnum orientale, Cibotium barometz, Dicranopteris linearis
var. linearis (Lai et al. 2009), Asplenium scolopendrium (Șuțan et al. 2016), etc.
were used to obtain extracts, which were subsequently tested for bioactivity. The
leaves of the edible species Stenochlaena palustris, at different ontogenetic stages
(young/mature sterile frond, young/mature fertile frond), were used by Chai et al.
(2012) to obtain extracts, which were characterized in terms of polyphenol content
and antioxidant activity. They proved to be a healthy source of food. Fern leaves are
a renewable resource, which may be used even when the species require
protection.
The antioxidant activity and the polyphenol content were tested for the
extracts obtained from rhizomes of Drynaria fortunei, Pseudodrynaria coro-
nans, Davallia divaricata, D. mariesii, D. solida and Humata griffithiana, spe-
cies that compose the Chinese traditional formula “Gusuibu” (Chang et al. 2007).
Rhizomes of Polypodium vulgare (Souri et al. 2008), Drynaria quercifolia
(Kandhasamy et al. 2008), Polypodium interjectum, Polystichum woronowii, P.
aculeatum, Asplenium scolopendrium, A. adiantum-nigrum, Dryopteris affinis,
Pteris cretica and Athyrium filix-femina were also used to obtain and character-
ize extracts (Valizadeh et al. 2015); some of the extracts were supplemented with
silver nanoparticles (Șuțan et al. 2016).
Spores, which are produced in large amounts by ferns, have constituted the bio-
logical material for some extracts, whose allergenic, carcinogenic and cytogenetic
effects were tested. In high biodiversity areas, pteridophyte spores are natural atmo-
spheric pollutants (Bunnag et al. 1989). These are found in large amounts in the
closed spaces of botanical gardens or in closed private spaces (Simán et al. 2000).
They may affect human health due to their allergenic or carcinogenic action. Thus,
the spores of the species Acrostichum aureum (Bunnag et al. 1989), Lycopodium
(Devi et al. 1989), Asplenium nidus, Dicranopteris curranii, D. linearis, Nephrolepis
auriculata, Pteridium aquilinum or Stenochlaena palustris (Chew et al. 2000) are
allergenic, while those of the species Anemia phyllitidis, Dicksonia antarctica,
Pteridium aquilinum, Pteris vittata and Sadleria pallida (Simán et al. 2000) are
known for their carcinogenic action. Furthermore, the extracts from spores of
Athyrium filix-femina were evaluated for their potential in the photosynthesis of
silver nanoparticles (AgNPs) and for their cytogenetic effects (see Subchap. 16.7).
The physical condition of the plant material used for obtaining extracts can influ-
ence their composition and bioactivity. For example, certain flavonoids can be
degraded when using fresh plant material (Marston and Hostettmann 2006). The
raw plant material necessary for the extracts was fresh, frozen or dried and pulver-
ized, as follows: fresh leaves of Athyrium filix-femina, Dryopteris affinis, D. filix-
mas (Soare et al. 2012b) and Asplenium nidus (Nath et al. 2013); fresh rhizomes of
Drynaria quercifolia (Kandhasamy et al. 2008); dried leaves of Asplenium
adiantum-nigrum, A. trichomanes (Hammami et al. 2016) and Stenochlaena palus-
tris (Chai et al. 2012); dried leaves and rhizomes of Polypodium interjectum,
Polystichum woronowii, P. aculeatum, Asplenium scolopendrium, A. adiantum-
nigrum, Dryopteris affinis, Pteris cretica and Athyrium filix-femina (Bahadori et al.
2015); dried rhizomes of Drynaria fortunei, Pseudodrynaria coronans, Davallia
divaricata, D. mariesii, D. solida and Humata griffithiana (Chang et al. 2007); and
frozen rhizomes and leaves (−18 °C) of Asplenium scolopendrium (Șuțan et al.
2016).
In some cases, the extracts were obtained from plant material pulverized in liquid
nitrogen. Thus, in order to determine the polyphenol content, and the antioxidant,
antibacterial and tyrosinase-inhibiting activity, fresh leaves of some medicinal
plants were pulverized in liquid nitrogen. The extraction was performed using meth-
anol, in a rotary orbital shaker (Lai et al. 2009).
Specialist literature mentions extracts obtained with the help of one or more
solvents. Diverse categories of solvents selectively extract the components from the
plant material, which may show different bioactivity. The solvents that are fre-
quently used to obtain crude extracts include water, ethanol, methanol, dichloro-
methane, petroleum ether, hexane, benzene, chloroform and acetone (Chai et al.
2012, 2015; Chang et al. 2007; Kandhasamy et al. 2008; Lai et al. 2009; Rajesh
et al. 2016; Roudsari et al. 2012; Souri et al. 2008; Valizadeh et al. 2015; Xie et al.
2015).
The evaluation of the bioactivity of crude extracts was either directly performed
on these extracts or after their concentration and fractionation. For example, crude
ethanol extracts were obtained from the dried and pulverized leaves of the species
Psilotum nudum, Nephrolepis biserrata and N. cordifolia. The extracts were con-
centrated in a rotary orbital shaker, freeze-dried and afterwards fractionated with
hexane, chloroform, ethanol and water (Rani et al. 2010). Each of the obtained frac-
tions was tested for antibacterial and antifungal properties.
16.3 A
ntimicrobial Activity of New Extracts
and Formulations
Pteridophytes contain a wide range of bioactive compounds, from the terpenoid,

phenolic, flavonoid and alkaloid classes (Ho et al. 2011). Alcaraz et al. (2000),
Cushnie and Lamb (2005) and Yusuf (1994) mentioned the presence of secretory
epidermal tissues on fern leaves and tissues that produce alkaloids, flavonoids, gly-
cosides and phenolic compounds, which are substances with antimicrobial proper-
ties. Flavaspidic acids PB and AB, isolated from the methanol extract obtained from
the rhizome of Dryopteris crassirhizoma, are responsible for the powerful antimi-
crobial activity against Staphylococcus aureus MRSA, Streptococcus mutans and
Bacillus subtilis (Lee et al. 2009). Eicosenes and heptadecanes have an antibacterial
and antifungal activity (Abraham and Kaushik 2015; Dalli et al. 2007). Recent
research showed that some phenolic compounds inhibit bacterial virulence factors
(T3SS-type III secretion systems) present in many Gram-negative pathogenic bac-
teria (Khokhani et al. 2013). It was demonstrated that fern extracts have antimicro-
bial effects on some Gram-negative and Gram-positive pathogenic bacteria,
including multidrug-resistant bacteria and on certain species of fungi.
Numerous research studies have emphasized the link between the antimicrobial
activity of the extracts and the solvents used to obtain them. Thus, Pal (2012)
observed that the most effective antimicrobial activity against E. coli was exhibited
by acetone extracts obtained from leaves of Athyrium filix-femina (inhibition zone/
IZ = 18 mm). Ethanol extracts produced an inhibition zone of 15 mm and methanol
extracts of 12 mm; the weakest activity was observed in the case of aqueous extracts
(IZ = 7 mm). Aqueous extracts from leaves of Psilotum nudum, Nephrolepis biser-
rata and N. cordifolia showed a more powerful antimicrobial activity against nine
bacterial strains (Rani et al. 2010) than the chloroform, hexane or ethanol extracts.
The extracts from the rhizome of Drynaria quercifolia obtained using ethanol,
methanol, petroleum ether, hexane, benzene or chloroform exhibited a different
antibacterial activity, dependent on the solvent. Thus, the diameter of the inhibition
zone produced by the extracts against Escherichia coli was 10 mm for the ethanol,
methanol and benzene extracts and 8 mm for the chloroform extract, while the
extracts for which petroleum ether and hexane had been used showed no antimicro-
bial activity (Kandhasamy et al. 2008).
The part of the plant used to obtain extracts influences their antimicrobial activ-
ity. Thus, for the methanol extracts from leaves of spontaneously occurring plants of
Polypodium vulgare and from gametophytes with sporelings (generated in vitro),
the antibacterial effects were different. The extract from leaves exhibited an inhibi-
tion zone of 12 mm against a bacterial strain isolated from the soil, while the extract
from the gametophyte with sporelings created an inhibition zone of 8 mm (Soare
et al. 2012a).
For multidrug-resistant bacteria, new antibacterial products can be developed,
starting from the pteridophyte extracts that have been tested. Aqueous, methanol
and chloroform extracts from the leaves of Lygodium flexuosum presented an
important antimicrobial activity against multidrug-resistant bacteria such as

methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
Enterococcus faecalis (VRE), as well as against other clinically isolated bacterial
strains (Nayak et al. 2013). The antibiotic activity of the extract, which, in some
cases, was more effective than that of the antibiotic used as a control sample, indi-
cates the fact that the plant is a source that must be taken into consideration and
further investigated for this property.
The antibacterial effects of pteridophyte extracts were evaluated for their effec-
tiveness against phytopathogenic bacteria. The fraction obtained with anhydrous
ethyl acetate from the methanol extract of Azolla microphylla inhibited the growth
of Xanthomonas oryzae (Abraham and Kaushik 2015), one of the most important
rice pathogens (Fun et al. 2017). The ethyl acetate fraction contains a mixture of
eicosenes and heptadecanes, compounds that were identified in the extract of Pteris
biaurita, an extract that was tested against the fungi Curvularia lunata, Fomes
lamaoensis, Poria hypobrumea, Fusarium oxysporum and against the bacterium
Bacillus pumilus (Dalli et al. 2007). The antifungal activity of pteridophyte extracts
was also emphasized for the following species: Saccharomyces cerevisiae,
Aspergillus niger (Soare et al. 2012b), Candida albicans (Amoroso et al. 2014),
Microsporum gypseum, Trichophyton mentagrophytes and T. rubrum (Rani et al.
2010), etc.
In human or animal organisms, the natural compounds ingested are metabolized
producing certain biological responses. As such, the results reached for a crude
extract, for whole fruits or vegetables may be different from those reached for com-
pounds isolated from that particular extract or for parts of fruits or vegetables (Lila
and Raskin 2005; Liu 2003).
A series of interactions, known as endointeractions, occurs among the existing
components of a plant extract (Lila and Raskin 2005); they produce the biological
response. The combination of a plant extract with another extract or with a synthetic
medical drug produces exointeractions (Lila and Raskin 2005), which modify the
biological response. These phytochemical interactions can explain the state of
health of people from a certain geographical area, known for a certain food behav-
iour (Lila and Raskin 2005).
Antimicrobial activity of pteridophyte extracts may be modified through combi-
nations with other extracts or other synthetic medical drugs, in new formulations
with therapeutic potential. The interactions that take place in such combinations can
be cummulative, if the effect obtained for the new formulation sums up the separate
effects. A particular interest is shown to those associations that produce synergetic
effects, significantly higher than the sum of the separate effects. It is possible for the
phytochemical interactions that take place in the new formulations to produce nega-
tive effects (antagonistic effects). In such cases, the activity of the active compounds
is inhibited (Lila 2009). In some other cases, the combination enhances bioavail-
ability, solubility and absorption of bioactive components (Eder and Mehnert 1998;
Gartner et al. 1997).
The research on the synergetic effect of the combination antibiotic plant product
has become a key instrument in phytomedicine (Santos et al. 2015). The combination
between curcumin and some synthetic antibiotics produced synergetic antibacterial

effects against Staphylococcus aureus (Teow and Ali 2015). The generation of
nanoparticles of the solid lipid (SLNs) type in formulations that contain ampicillin
and curcumim determined a decrease in the MIC (minimum inhibitory concentra-
tion) and MBC (minimum bactericidal concentration) as compared to the formula-
tion that contains only ampicillin and curcumin, a result that also suggests a decrease
in the risk of developing resistance to antibiotics (Alihosseini et al. 2016). Green tea
polyphenols, combined with oxacillin, produce a decrease in the MIC of the antibi-
otic in the fight against Staphylococcus aureus (MRSA) (Yoshida et al. 2005).
Formulations that contain kaempferol isolated from Impatiens balsamina and
erythromycin or clindamycin showed synergetic antibacterial effects against
Propionibacterium acnes (Lim et al. 2007). Different flavonoids and carotenoids in
combination with antibiotics also led to synergetic antibacterial effects against
Pseudomonas aeruginosa (Santos et al. 2016). The methanol extract of Punica gra-
natum, combined with the antibiotics chloramphenicol, gentamicin, ampicillin, tet-
racycline and oxacillin, has a synergetic effect against methicillin-resistant
Staphylococcus aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) (Braga
et al. 2005).
Pteridophyte extracts have been considered in the evaluation of the possible
enhancing antibacterial effects produced in combination with antibiotics. New com-
binations between methanol extracts from leaves of Dryopteris filix-mas (D) and the
antibiotics piperacillin (Pip) and tetracycline (Te), respectively, produced larger
inhibition zones than those corresponding to the antibiotic or the extract when used
independently. The new combinations were created by impregnating standard anti-
biotic discs with the extract. The formulation D + Te produced better results against
Staphylococcus aureus and Enterobacter cloacae and the formulation D + Pip
determined larger inhibition zones against S. aureus, Enterobacter cloacae and a
bacterial strain isolated from the soil (Soare et al. 2012c). Similar results were
reported by Parihar et al. (2006) for the aqueous and alcoholic extracts from leaves,
rhizomes and roots of Athyrium pectinatum. Thus, a mixture of equal parts of extract
and antibiotic may show, in some cases, greater effectiveness than the antibiotic
alone.
16.4 S
ynthesis of “Green” Nanoparticles Using Pteridophyte
Extracts
The therapeutic use of pteridophytes has had a spectacular evolution, starting from
their use in the traditional medicine of different peoples to the current stage in which
pteridophytes are used in the form of nanoparticles.
The nanotechnology industry was a global business evaluated at about 4 million
dollars about 10 years ago and 1 billion dollars in 2015 (Roco 2005). At present,
hundreds of products containing biomaterials have applicability and are used on a
large scale in the fields of electronics, optics, in the process of food packaging, in
medicine and the cosmetic industry, in wastewater treatment technology or in envi-
ronmental remediation processes (Aitken et al. 2006; Nowack and Bucheli 2007;
Handy et al. 2008a; Klain et al. 2008). A few thousand other nanomaterials are
being researched at the moment.
Nanoparticles are the smallest increments of the physical world; they are of simi-
lar scale to the biomolecules that govern the existence of life. The DNA molecule
has a diameter of approximately 2 nm, while the C60 diameter is approximately
5 nm. These structures, as a result of their nanometric dimensions (variable between
1 and 100 nm), have some modified characteristics (resistance, conductivity, cata-
lytic reactivity, magnetic or optical properties), leading to a collateral interpretation
of the common laws of science (Handy et al. 2008b). The nanoparticle behaviour
and its potential side or favourable effects on the living systems do not depend only
on the dosage but also on their surface, size and shape. These effects are also based
on their physical and chemical properties. For example, the solubility of otherwise
insoluble substances may increase dramatically when the size of their particles is
smaller than 100 nm. Furthermore, nanoparticles have a larger surface compared to
the particles with a similar mass, which leads to a higher surface reactivity (Auffan
et al. 2009). Surfactants and other additives can modify the surface characteristics
of nanoparticles and can prevent their aggregation (European Comission 2006). The
similarity between natural biomolecules and designed nanomaterials may be trans-
lated into an enormous potential of the latter to interfere in biological processes.
These interactions might affect the cell membrane behaviour and biochemical pro-
cesses or even the genetic code itself (Klaine et al. 2012).
In the last decade, the elaboration of one-step methods for the synthesis of metal
nanoparticles has been stimulated by their multiple practical applications. Even
though the synthesis of metal nanoparticles may be induced through various physi-
cochemical methods, the biogenic reduction of metal ions is a rapid process, which
can be performed at room temperature and pressure, with increased intensity. At the
same time, nanoparticle biosynthesis, as a cost-effective and nontoxic method, safe
to the environment, is preferable when the finite product is to be used in biomedical
and pharmaceutical applications (Mittal et al. 2013). Nanoparticles offer the advan-
tage that they can be encapsulated and released in a controlled and targeted
manner.
Diverse biological systems, such as plants, algae, bacteria, yeasts and fungi, were
proved to be excellent bioreactors for the in vivo synthesis of nanoparticles, but the
use of plant extracts, combined with particular acids or some metal salts, such as
silver (Ag), gold (Au), copper (Cu), platinum (Pt), zinc (Zn), cesium (Cs), titanium
(Ti), indium (In) salts and many others, offer the advantage of an effective control
over nanoparticle biosynthesis and purification.
Specialist literature indicates a relatively recent date for the first nanoparticle
synthesis using fern extracts. Kang et al. (2008), using Pteridophyta, reported the
synthesis of silver nanoparticles (AgNPs) by means of “green chemistry”; these are
the most popular types of nanoparticles encountered in biosynthesis methods. Later
on, extracts from Actiniopteris radiata were used to reduce silver ions to
n anoparticles with a diameter ranging from 20.8 nm to 30.6 nm, while AgNPs
measuring between 35 and 65 nm were synthesized in extracts from Adiantum
capillus-veneris. Silver nanoparticles, whose synthesis was mediated by the extract
from Nephrolepis exaltata, without supplementing the reaction medium with
capping and stabilizing agents, were characterized as polydispersed, with a large
amplitude of their mean size between 22 and 44 nm (Raut et al. 2010 cited by
Chrislyn et al. 2016).
Analyses performed by means of transmission electronic microscope (TEM) and
selected area electron diffraction (SAED) showed that gold and silver nanoparticles
photosynthesized through the reduction of tetrachloroauric acid and AgNO3 in the
extract of Adiantum philippense, respectively, were monocrystalline and anisotropic
and had variable sizes, between 10 and 18 nm (Sant et al. 2013). It was appreciated
that the reduced sizes of these NPs, approximately 11 nm for AuNPs and about
13 nm for AgNPs, were due to the alkaline pH value and the photosynthetic environ-
ment. The same method was applied to the Au ions which were reduced to nanopar-
ticles with an average size of 8.3 nm in the extract from Azolla microphylla. The
shape of Ag and Au nanoparticles, whose synthesis was mediated by fern extracts,
was predominantly spherical, but the HRTEM analysis with higher magnification
indicated that they were spherical, triangular, hexagonal and rod shaped (Kunjiappan
et al. 2015).
16.5 T
he Role of Metabolites from the Pteridophyte Extracts
in Nanoparticle Synthesis
For nanoparticle biosynthesis, fern extract is mixed with a metal salt at room tem-
perature. The reaction is completed in a few minutes to a few hours. The molecular
basis for the biosynthesis of AgNPs is not known, but it has been speculated that the
organic matrix favours the bonds between Ag and proteins, through the aminoacid
fragments that serve as nucleation sites.
Most often metal nanoparticles have been produced by condensation, a principle
that was proposed by Turkevich et al. (1951). According to Balaji et al. (2008),
proteins/enzymes are responsible for reducing metal ions when plant extracts are
used in nanoparticle synthesis. Polysaccharides, polyols, heterocyclic compounds
soluble in water and, generally, phytochemical compounds with reductive or anti-
oxidant properties are recognized as the main agents responsible for reducing metal
compounds and for stabilizing nanoparticles, respectively (Huang et al. 2007;
Geethalakshmi and Sarada 2010; Prathna et al. 2010; Ghosh et al. 2011, 2012a, b;
Park et al. 2011).
Phytochemical screening of fern extracts showed their content of proteins and
secondary metabolites, such as phenols, alkaloids, tannins, flavonoids, carbohy-
drates, saponins, glycosides, steroids and terpenoids (Pan et al. 2011; Panneerselvam
et al. 2016; Xavier et al. 2016), to which the reductive and stabilizing properties are
attributed.
For example, the FTIR (Fourier-transform infrared spectroscopy) analysis of the
extracts from A. capillus-veneris and Cyathea nilgerensis demonstrated phenols,
when combined with AgNPs, prevent their agglomeration (Samidoss et al. 2013;
Johnson et al. 2017). For the extracts from Pteris tripartita, flavonoids were men-
tioned to have an extremely important role in reducing the Ag ions. The implication
of phenols, as well as that of aliphatic amines, alcohols, amides and carbonyl groups
from biomolecules, was also suggested in the process of stabilization of silver
nanoparticles in the extracts from Dicranopteris linearis (Rajaganesh et al. 2016;
Baskaran et al. 2016).
The reductive capacity of metal ions was tested for different types of extracts,
such as the fresh aqueous extracts from dried plants of Actiniopteris radiata (Sant
et al. 2013) or from plants/green leaves of Adiantum capillus-veneris (Santhoshkumar
and Nagarajan 2014), A. philippense (Sant et al. 2013) and Azolla pinnata
(Korbekandi et al. 2014); boiled aqueous extracts from fresh leaves of Cheilanthes
farinosa (Nalwade et al. 2013), Nephrolepis exaltata (Chrislyn et al. 2016),
Dicranopteris linearis (Rajaganesh et al. 2016), Pteris argyraea, P. confusa, P.
biaurita (Britto et al. 2014), P. tripartita (Baskaran et al. 2016) and Nephrolepis
exaltata (Bhor et al. 2014); crude methanol extracts from dried plants of Azolla
microphylla (Kunjiappan et al. 2015) or extracts concentrated by evaporation from
dried plants of Cyclosorus interruptus, Christella dentata and Nephrolepis cordifo-
lia (Xavier et al. 2016); and crude ethanol extracts from leaves or rhizomes of
Asplenium scolopendrium (Şuţan et al. 2016) or Dryopteris crassirhizoma (Lee
et al. 2016).
The antioxidant potential and the medicinal value of fern species have been the
determining factors in selecting them for the synthesis of Ag and Au nanoparticles.
At the same time, the quantitative and qualitative diversity of the bioactive ingredi-
ents from fern extracts may be an explanation for the morphological diversity of
nanoparticles (Makarov et al. 2014).
The bioactive compounds found in the composition of the extracts, the solvent
used for extraction, the concentration of the extract and that of the metal salt, the
electrochemical potential of metal ions, the pH of the reaction mixture, the incuba-
tion period and the period of contact were often mentioned as influential factors in
the nanoparticle production rate, as well as in the resulting characteristics.
The first indication of the biosynthesis of AgNPs is given by the change in the
colour of the reaction mixture. For example, in only 20 s, the phytosynthesis of
AgNPs induced by the extract from Salvinia molesta determined a colour change in
the reaction mixture from green yellowish to reddish brown (Verma et al. 2016).
The addition of the extract from Adiantum philippense to the solution of tetrachlo-
roauric acid (HAuCl4·3H2O 1 mM) determined a colour change in the mixture from
greenish to dark blue (Sant et al. 2013). In the case of the extract from Azolla micro-
phylla, through the biosynthesis of AuNPs, the reaction mixture turned dark pink
(Kunjiappan et al. 2015).
The pH value of the reaction mixture has an extraordinary influence on the

nanoparticle formation process. First, pH modification is correlated with quantita-
tive and qualitative diversification of phytochemical compounds contained in the
extract, as well as with the variation of their potential for binding and reducing
metal cations and anions during nanoparticle synthesis, determining changes in
shape, size and synthesis yield. For example, Chrislyn et al. (2016) showed that the
synthesis speed of AgNPs in extracts from Nephrolepis exaltata was higher than in
the alkaline medium. Similarly, the UV-Vis spectrum confirmed that pH = 11 was
optimal for the synthesis of AuNPs. AgNPs were best synthesized when pH = 12,
for the synthesis assisted by the extract from Adiantum philippense (Sant et al.
2013). However, the same authors pointed out that for a lower pH, the absorption
capacity was lower and wider. These findings confirm the results according to which
the alkaline pH stabilizes and prevents the agglomeration of synthesized
nanoparticles.
The size of the phytosynthesized NPs varied depending on the reaction medium.
Thus, AgNPs whose biosynthesis was mediated by the extract from Cyathea nilge-
rensis varied on average between 45 nm and 74 nm (Johnson et al. 2017). The small-
est AgNPs with a diameter of 0.93 nm and a mean size of 6.5 nm were obtained in
the biosynthesis reaction catalysed by the extract from Azolla pinnata (Korbekandi
et al. 2014).
Kinetic studies revealed that temperature is a decisive factor in the process of
nanoparticle biosynthesis. For example, the reaction rate and biosynthesis efficiency
of anisotropic Au and Ag nanoparticles (spherical, triangular and decahedral) in
extracts from Diplazium esculentum (Sinha and Ahmaruzzaman 2016) were
favoured by the increase in temperature. It is estimated that the increase in tempera-
ture leads to the increase in the nucleation rate.
The concentration of the plant extract, as well as the concentration of metal salts,
is a defining parameter for the reaction of nanoparticle phytosynthesis. Thus, Sant
et al. (2013) showed that the undiluted aqueous extract from Adiantum philippense
had induced an effective synthesis of the Au and Ag nanoparticles, while the diluted
sample had displayed no significant nanoparticle synthesis. Moreover, mixing the
plant extract with tetrachloroauric acid/silver nitrate in a higher concentration (from
1 mM to 10 mM) induced nanoparticle synthesis at higher rates. In the case of the
tetrachloroauric acid, the increase in the biosynthesis rate was recorded up to a con-
centration of 5 mM; the increase in the concentration of metal ions resulted in a
decrease of biosynthesis intensity. For silver nitrate the optimal concentration of
metal ions was 9 mM.
In this context, it should be mentioned that the efficiency of metal nanoparticle
synthesis depends on the electrochemical potential of the ions, so that the ability of
plant extracts to reduce metal ions can be significantly higher in the case of ions
with a higher electrochemical potential (Ag+) compared to the ions with a lower
electrochemical potential (e.g. [Ag(S2O3)2]3−) (Haverkamp and Marshall 2009, cited
by Makarov et al. 2014).
16.6 A
pplications of Nanoparticles Phytosynthesized
in Pteridophyte Extracts
Up to the present, numerous studies have promoted fern extracts as renewable

sources, safe to the environment, which can be successfully used to reduce metal
ions to bioparticles. In spite of the fact that nanoparticles biosynthesized in plant
extracts are of real interest, till now, they have been tested in a relatively low number
of in vitro or in vivo practical applications for their antioxidant, antimicrobial, anti-
inflammatory, larvicidal and pupicidal activity. For example, the DPPH assay, fer-
rozine and phosphomolybdenum assay and ABTS+ assay revealed a significant
difference in the antioxidant activity of biosynthesized AgNPs when compared to
the aqueous extract from Pteris tripartita (Baskaran et al. 2016). AuNPs biosynthe-
sized in extracts from Azolla microphylla were shown to have a significant antioxi-
dant capacity as well as a liver protective activity. They were promoted as potential
protection and prevention agents against liver diseases (Kunjiappan et al. 2015).
Comprehensive studies on the in vitro antimicrobial activity of AgNPs biosyn-
thesized using fern extracts demonstrated their powerful or moderate antimicrobial
effect against certain pathogenic agents. It is considered that antimicrobial activity
is dependent on the size and dosage of AgNPs and that their inhibitory activity is
more effective against Gram-positive bacteria than against Gram-negative bacteria
(Koteswaramma et al. 2017).
Silver nanoparticles whose synthesis was mediated by extracts from Nephrolepis
exaltata exhibited an important antimicrobial activity as compared to those that
were chemically synthesized, which presented either no antimicrobial activity or an
antimicrobial activity comparable to that of standard antibacterial agents (Bhor
et al. 2014; Chrislyn et al. 2016). AgNPs phytosynthesized in extracts from
Cheilanthes farinosa induced an inhibition zone of 15 mm against Staphylococcus
aureus and 20 mm against Proteus morganii, an activity similar to that manifested
by tetracycline (Nalwade et al. 2013). In addition, Kalita et al. (2016) underlined the
biocompatibility and functionalization of the AuNPs surface biosynthesized using
aqueous extracts from Adiantum philippense with the broad-spectrum antibiotic
beta lactam (amoxicillin). AuNPs combined with amoxicillin enhanced the in vivo
and in vitro bactericidal activity against Gram-positive and Gram-negative
bacteria.
The action of AgNPs and Ag2+ was reported to be similar, although nanoparticles
proved to be more efficient at much lower concentrations than ions. The exact
mechanism of their antibacterial activity has not been discovered yet, but some
researchers claim that AgNPs attach to the cell membrane, disrupt its permeability
and determine structural modifications of the cell membrane, eventually leading to
the death of the cell (Krishnaraj et al. 2012).
Morones et al. (2005) and Baker et al. (2005) suggested that AgNPs may enter
bacteria and fungi producing damage due to their interaction with phosphorus and
sulphur electrons from compounds such as DNA and proteins.
Several studies have evaluated the mosquitocidal effect of nanoparticles whose

synthesis was induced in pteridophyte extracts. Based on the findings resulted from
the experiments focused on the ovicidal properties and especially on the oviposition
deterrent effects of AgNPs synthesized using extracts from Dicranopteris linearis,
Rajaganesh et al. (2016) recommended them particularly for the integrated control
programs against arbovirus vectors.
Other studies emphasized the larvicidal and pupicidal potential of aqueous
extracts from Adiantum capillus-veneris supplemented with AgNPs against the vec-
tor of malaria, Anopheles stephensi (Samidoss et al. 2013), or that of the extracts
from Cyclosorus interruptus, Christella dentata and Nephrolepis cordifolia against
the pest Spodoptera litura (Xavier et al. 2016). The same authors argued in favour
of the use of extracts of Nephrolepis exaltata with AgNPs in the treatment of waste-
water contaminated by Escherichia coli.
Gold and silver nanoparticles biosynthesized in extracts from Diplazium escul-
entum were tested for their catalytic properties in the remediation of two carcino-
genic textile dyes (methyl violet 6B and rose bengal) and of the phenolic compound
4-nitrophenol. The depleted nanostructured materials and the intermediary products
of the degradation process, along with the period of dye degradation by the nano-
structured catalysts, recommend them as ideal candidates in wastewater treatment
processes (Sinha and Ahmaruzzaman 2016).
The interest shown to nanoparticles and nanomaterials must be supported by
empirical research (Bernhardt et al. 2010; Klaine et al. 2012) and scientific argu-
ments that reduce fear and concern about potential side effects of nanotechnological
products on human and natural ecosystems. The vast majority of the research con-
ducted so far has tested the exposure of living systems to much higher nanoparticle
concentrations (mg L−1) than the evaluated natural risks (μg L−1). Appreciating and
defining the dosage-response curve in correlation with reality is essential, especially
because most scientific results suggest that nanomaterials have a chronic action at
low concentrations (Gottschalk et al. 2009). The determination of the correct dos-
age, validated by reality, must be followed by the creation of a scenario that includes
the most efficient way to introduce nanoparticles in the experimental system and the
evaluation of transportation, targets as well as the nanoparticle physicochemical
changes during the experiment.
16.7 E
valuating Phytotoxicity and Cytogenetic Effects
of the Ethanol Extracts from Spores of Athyrium filix-
femina (L.) Roth
A considerable number of species, including bacteria (Kumar et al. 2011; Wang
et al. 2011), algae (Manzo et al. 2013), plants (Makarov et al. 2014; Allafchian et al.
2016; Jeevanandam et al. 2017), invertebrates (Croteau et al. 2014) and vertebrates
as well as fish (Ma et al. 2013) and rats (Magaye et al. 2014) have been the subject
of many nanotoxicological studies.
Despite the fact that numerous fern species have been largely used as natural
remedies for hundreds of years, at the moment there are relatively few data regard-
ing the phytotoxicity and cytogenotoxicity of fern extracts. Using the cytokinesis-
block micronucleus cytome assay, Glamočlija et al. (2014) remarked that the
cytotoxic and genotoxic potential of the Asplenium scolopendrium extracts on
in vitro human lymphocytes was very low and null, respectively.
The extracts from spores of Anemia phyllitidis, Dicksonia antarctica, Pteridium
aquilinum, Pteris vittata and Sadleria pallida induced in vitro DNA damage (Simán
et al. 2000). Chai et al. (2015) evaluated the cytotoxicity of aqueous extracts from
Christella arida, C. dentata, Cyclosorus interruptus, Microsorum punctatum,
Nephrolepis acutifolia and Pleocnemia irregularis on leukaemia cell line (K562),
emphasizing their therapeutic effects. Lok et al. (2007) concluded that AgNPs do
not have a direct effect on DNA or proteins.
In our research aimed to determine the cytogenetic effects of extracts obtained
from spores of Athyrium filix-femina (L.) Roth, we used the test organism Allium
cepa L. (2n = 16). The Allium test provides data that may be correlated with the
results obtained for the prokaryotic systems (Fiskesjö 1985). The spores were col-
lected from plants found in the Vâlsan Valley, Argeș, Romania, at the following
location: N45°20′18″, E0.24°44′04.7″, at an altitude of 730 m. The ethanol extracts
were prepared through spore mass suspension in ethanol (1:100 w/v). The mixture
was kept for 48 h at room temperature. The extracts obtained (EEA) were filtered
using filter paper (Whatman no. 1) and the filtrates were used for nanoparticle phy-
tosynthesis and for evaluation of the cytogenotoxic potential using the Allium test.
The synthesis of AgNPs was induced by treating 100 ml of ethanol extract from
spores of A. filix-femina with 100 ml of aqueous solution of AgNO3 1 mM and by
incubating the mixture in the dark, at room temperature for 3 h.
A HITACHI SU8230 cold-field emission scanning electron microscope was used
for scanning electron microscope analysis (SEM). Before subjected to analysis,
plant extract solution was dropped and dried on scanning transmission electron
microscopy (STEM) copper (Cu) grids. A secondary electrons detector (SE) was
used for sample morphology and particle size measurements. For the energy disper-
sive X-ray spectroscopy analysis (EDS), several point scans were performed on
relevant sample spots on each sample. Because of sample charging at higher volt-
age, quantitative EDS analysis could not be properly conducted. Still, qualitative
EDS analysis revealed the presence of Ag. Additionally, for spore extract supple-
mented with AgNPs in a concentration of 25%, an EDS-mapping analysis was per-
formed in order to observe Ag distribution on the analysed sample surface.
The cytogenetic effects of the extracts, before and after Ag nanoparticle phyto-
synthesis, were evaluated considering the modifications in mitotic index and the
stages of mitotic division (prophase, metaphase, anaphase and telophase), as well as
the frequency of chromosomal aberrations and nuclear anomalies induced in the
meristematic root cells of Allium cepa L. (Șuțan et al. 2016). The evaluation of the
cytogenetic effects consisted in a 96 h exposure of the bulbs, initially to the action
Fig. 16.1 Colour change in the extract from spores of Athyrium filix-femina (L.) Roth after adding
AgNO3 (left, before the addition of AgNO3; right, after the formation of nanoparticles)
of distilled water (for 48 h) and of some varied concentrations of ethanol extracts
from spores, without (EEA 5%, 15% and 25%) and with AgNPs (EEA + AgNPs
5%, 15% and 25%).
The microscopic preparations were analysed with an Olympus CX-31 micro-
scope, using a ×400 objective. The microscopic analysis consisted in determining
the number of cells found in different phases of mitosis and the frequency of chro-
mosomal and nuclear aberrations reported for a number of 3000 cells for each
experimental sample. The mitotic index was calculated as the percentage ratio of the
number of cells in the mitosis process to the total number of analysed cells (Tedesco
and Laughinghouse 2012). The percentage ratio of cells in prophase, metaphase,
anaphase or telophase was determined based on the total number of cells in mitosis.
The frequency of chromosomal aberrations and nuclear anomalies was determined
by relating them to the adequate phase of the cell and mitotic cycle.
The experiments were performed in triplicate, and 3000 cells were analysed for
each experimental sample. Statistical analysis of results was conducted using the
IBM SPSS Statistics 20 program (2011). Statistical significance and significant dif-
ferences between variables were determined using variance analysis (one way
ANOVA) and Duncan’s test for multiple comparison, respectively. The values
P ≤ 0.05 were considered statistically significant. Graphs and tables were compiled
based on the mean values ± standard error resulted from several independent
experiments.
The formation of Ag nanoparticles in extracts from spores of A. filix-femina was
established by visual observation of colour change from light yellowish (EEA) to
light brown (EEA + AgNPs), as shown in Fig. 16.1. SEM analysis confirmed
nanoparticle phytosynthesis (Fig. 16.2). The presence of silicon, according to EDS
results (Fig. 16.3), is not surprising, considering the fact that silicon was mentioned
in the atomic composition of Selaginella spores (Tryon and Lugardon 1978) and
that of the leaves of some fern species (Höhne and Richter 1981). Aluminium (Al)
presence on EDS spectra is due to the sample holder interference, while the copper
(Cu) signal is due to the STEM grid on which the plant extract solution was dropped
and dried. Thus, the presence of Cu and Al on sample EDS spectra is not relevant
Fig. 16.2 SEM analysis of AgNPs phytosynthesized in extracts from spores of Athyrium
filix-femina (L.) Roth
Fig. 16.3 EDS analysis of extracts from spores of Athyrium filix-femina (L.) Roth supplemented
with AgNPs
for the chemical elemental composition of the sample and should be excluded from
EDS analysis.
Root length was the macroscopic parameter taken into account in the evaluation
of extract toxicity. For all experimental samples, root length was measured after
72 h, and the results were compared to the negative control sample. Analysis of
results indicated growth inhibition in the roots of A. cepa exposed to different con-
centrations of extracts from spores of A. filix-femina, with and without AgNPs
(Fig. 16.4).
The roots of A. cepa had an average length of 21.46 mm in the experimental
control sample, while the roots exposed to treatments with extracts from spores
reached an average of 16 mm, which was the length of the experimental sample
(EEAs 5%). Statistical analysis of results showed a statistically significant correla-
tion between root length and extract concentration. However, the results confirmed
the stimulating effect of the extracts, compared to the samples defined by the
corresponding ethanol concentrations for root growth. Improved root tolerance to
30.00
Average root lengths (mm) 25.00
20.00
b
15.00 c
d
10.00 def de
def
ef ef
f
5.00
0.00
5%
%
%
l
5%
%
5%
EE %
tro
15
25
l5
15
25
5
PS
on
l1
l2
As
PS
PS
no
As
As
N
C
no
no
ha
N
Ag
EE
EE
Ag
Ag
ha
ha
Et
s+
Et
Et
+
A
As
As
EE
EE
EE
Fig. 16.4 The influence of the extract from spores of Athyrium filix-femina (L.) Roth on root
length of Allium cepa L. before and after phytosynthesis of AgNPs (Data are shown as mean values
± SE of three replicates; a, b, c, d, e, f – interpretation of statistical significance and significant
differences through Duncan’s test, p <0.05)
low water concentration can be attributed to the presence of silicon (Eneji et al. 2005;
Hattori et al. 2005) identified in the spore extracts through EDS analysis.
According to Fiskesjö (1993), the growth of the roots is inhibited when they are
exposed to the action of toxic substances, to an inappropriate pH value or to undis-
solved substances that limit their intake of nutrients. In our study, compared to the
negative control sample and to the spore extracts, the incubation of the roots in
extracts supplemented with AgNPs induced a significant growth delay, up to the
maximum mean value of 8 mm (EEAs + AgNPs 5%), and minimum mean value of
5 mm (EEAs + AgNPs 25%), values that can be attributed to the action of AgNPs.
Cytogenetic Effects of Spore Extracts Analyses of mitotic index variation, distribu-
tion of the phases of mitotic division, types of chromosomal aberrations and their
frequency were conducted to evaluate the cytogenotoxic effects of extracts from
spores of A. filix-femina, before and after phytosynthesis of AgNPs.
The variation of the mitotic index was one of the dependent factors considered
when analyzing variance and determining the significant differences between
experiments. The results are presented in Fig. 16.5. Statistical interpretation of the
microscopic results underlined a significant reduction in the frequency of cells in
mitosis in the meristematic root tips incubated in extracts from spores of A. filix-
femina compared to the negative control sample, for a confidence interval of 95%.
The MI value determined for the negative control sample was 10.65%, significantly
higher than the values presented by all the other experimental samples. Campos
et al. (2008) explained the mitodepressive effects of the extracts of Dicranopteris
12.00 a
10.00 b b b
% of diviving cells
8.00
c c c
d
6.00
c
4.00 c
2.00
0.00
%
5%
l
5%
%
5%
%
5%
tro
l5
25
15
25
15
l1
on
l2
no
PS
As
As
PS
PS
no
As
C
no
ha
EE
N
EE
ha
EE
N
ha
Ag
Et
Ag
Ag
Et
+
Et
+
As
As
As
EE
EE
EE
Fig. 16.5 Influence of the extract from spores of Athyrium filix-femina (L.) Roth on the mitotic
index in meristematic root cells of Allium cepa L. before and after phytosynthesis of AgNPs (Data
are shown as mean values ± SE of three replicates; a, b, c, d – interpretation of statistical signifi-
cance and significant differences through Duncan’s test, p <0.05)
flexuosa and Gleicheniella pectinata through their allelopathic action on meriste-

matic cells of Lactuca sativa and Zea mays.
The mitodepressive effects of spore extracts were independent of the extract con-
centration. In this context, we must highlight the stimulating effects of spore extracts
compared to the corresponding concentrations of ethanol. At the same time, statisti-
cal analysis of results revealed that the mitoinhibitory effects of spore extracts sup-
plemented with AgNPs were more powerful than those of extracts without
nanoparticles, which were comparable with the mitodepressive effect of ethanol.
The Mitotic Phase Distribution The analysis of the results regarding the distribu-
tion of mitotic phases is presented in Fig. 16.6. Statistical interpretation of data
emphasized a substantial prophase accumulation (98.83%) in the meristematic root
tips incubated in EEAs 5%. The increase in the concentration of spore extracts to
15% and 25%, respectively, did not generate major changes when compared to the
negative control sample. Ethanol treatments, as well as those with spore extracts
enriched with AgNPs, induced an important accumulation of metaphases and telo-
phases in the onion root tips.
The mitotic index is a solid parameter in cytogenotoxicity evaluation (Fiskesjö
1985; Fernandez et al. 2007). The reduction in the mitotic index can be explained by
mitotic cell cycle arrest, either in one of the stages of the interphase, through inhibi-
tion of nuclear proteins or DNA synthesis, or in one of the stages of mitosis, espe-
cially during the metaphase. In our study, the important increase in the frequency of
prophases after incubation of roots in spore extracts for 24 h suggested that a higher
number had passed the G2 phase. On the other hand, root treatment with spore
100% op
op lmnop
mnop hijkl lmnop
klmno jklmn nop lmnop hijkl hijk hij
90% ijklmn
klmnop klmno hijklm jklmn hijklm
lmnop lmnop lmnop
80% jklmno
Distribution of mitotic phases (%)
kl mnop f
hijkl
70% hi
fg ghi
60% ghi
50%
b b b
40%
a d c
30% cd
de de
e
20%
10%
0%
%
%
5%
%
l
5%
tro
5%
15
25
15
25
15
25
on
l
no
Ps
As
l
Ps
Ps
no
no
As
As
C
ha
N
EE
ha
ha
N
EE
EE
Ag
Et
Ag
Ag
Et
Et
+
As
As
As
EE
EE
EE
Telophase Anaphase Metaphase Prophase
Fig. 16.6 Influence of the extract from spores of Athyrium filix-femina (L.) Roth, with and without
AgNPs, on the distribution of mitotic phases in the meristematic root cells of Allium cepa L. (Data
shown as mean values ± SE of three replicates; a, b, c, d, e, f, g, h, i, j, k, l, m, n, o, p – interpretation
of statistical significance and significant differences through Duncan’s test, p <0.05)
extracts supplemented with AgNPs through biosynthesis induced the accumulation

of metaphases, probably as a consequence of a blockage in the formation/function-
ing of the mitotic spindle. This explanation is supported by the presence of C-mitoses
and polyploid cells, which were observed in the experimental samples
EEAs + AgNPs. It is estimated that some disrupting agents could prevent the attach-
ment of micronuclei to kinetochores, so that, following the signals generated by
control points, the transition from the metaphase to the anaphase is blocked.
However, prolonged treatments with disrupting agents help the mitotic checkpoint
to adapt, allowing the cell to complete the mitotic cycle by forming tetraploid cells
and cytokinesis (Jordan et al. 1991, cited by Blajeski et al. 2002). Genotoxic stress
activates intracellular signaling molecules, which leads to cell growth arrest, DNA
repair and/or apoptosis.
Chromosomal and Nuclear Aberrations Microscopic analysis of temporary squash
preparations displayed different types of chromosomal and nuclear aberrations,
such as laggards and vagrant chromosomes, sticky chromosomes, C-mitoses, binu-
cleate cells, variation of chromosomes number or budding nuclei, which character-
ized the various experimental samples (Fig. 16.7). Thus, laggards and vagrant
chromosomes were the most frequent chromosomal aberrations. Vagrant chromo-
somes were identified in all experimental samples, without major variations, while
Fig. 16.7 Chromosomal aberrations and nuclear anomalies identified in meristematic root cells of
Allium cepa L. that underwent treatment with spore extracts of Athyrium filix-femina (L.) Roth. ex
Mert. (a) vagrant chromosomes, EEAs 5%; (b) stickiness, ethanol 15%; (c) anaphase bridges,
EEAs + AgNPs 15%; (d) C-mitosis, EEAs + AgNPs 5%; (e) binucleate cell, EEAs 25%; (f) telo-
phase bridge, EEAs + AgNPs 15%; (g) variation of chromosome number, EEAs + AgNPs 25%;
(h) micronucleus, EEAs + AgNPs 25%; (i) nucleoplasmic bridges, EEAs + AgNPs 25%; (a–i): at
a magnification 400×
the laggards had the highest frequency in the meristematic root cells incubated in
ethanol 25% and a significantly lower frequency in the experimental samples
EEAs + AgNPs, independent of their concentrations. Spore extracts from A. filix-
femina induced a lower frequency formation of binuclear cells, while root incuba-
tion in spore extracts supplemented with AgNPs generated the formation of
C-mitoses and their accumulation with a frequency ranging from 8.33% up to
19.44% (Table 16.1). These observations revealed the different influence of spore
extracts on the mitotic apparatus, based on the presence or the absence of AgNPs.
With no correspondence between the aberration frequency and the extract con-
centration, the influence of bioactive compounds on cells in the division process is
obvious. McFee and Tice (1990) appreciated that a series of factors, such as com-
pound solubility, transport rate and biodistribution and the concentration at the tar-
get site, can modulate the time interval in which aberrations can be induced.
According to Rank (2003), the presence of vagrant chromosomes was an indica-
tor of the toxic effect on the mitotic spindle. Furthermore, the formation of abnor-
mal anaphases and the presence of C-mitoses were evidence of the side effects of
AgNPs on the spindle. The detection of binucleate cells in meristematic cells of
Table 16.1 Chromosomal aberrations and nuclear anomalies observed in the meristematic root cells of Allium cepa L. incubated in ethanol extracts from
spores of Athyrium filix-femina (L.) Roth, before and after phytosynthesis of AgNPs
Chromosomal aberrations Nuclear anomalies
Experimental Variation of
variants Vagrants Laggards Stickiness C-mitosis Binucleate cell chromosomes number Nuclear buds Others
Control 1.85 ± 1.85ab – – – – – –
Ethanol 5% 10 ± 5.77ab – – 10 ± 5.77ab – – – –
Ethanol 15% 20 ± 11.55ab 20.69 ± 14.98ab 4.99 ± 2.54ab 8 ± 0.04b – – – 0.04 ± 0.04b
Ethanol 25% 25.5 ± 12.8ab 30 ± 17.32a 22.8 ± 4.28ab 12 ± 0.3ab – – – 0.04 ± 0.04b
EEAs 5% 6.61 ± 4.15ab – – – 0.76 ± 0.20b – – 0.02 ± 0.02b
EEAs 15% 8.79 ± 8.79ab 21.42 ± 5.99ab 2.38 ± 2.38ab – 0.18 ± 0.1b – – 0.08 ± 0.08b
EEAs 25% 10.18 ± 3.03ab 13.88 ± 10.01ab 8.33 ± 4.80ab – 0.24 ± 0.08b – – 0.08 ± 0.04b
EEAs + AgNPs 2.22 ± 2.22ab 0.66 ± 0.66b – 8.33 ± 8.33ab – 0.05 ± 0.05b – –
5%
EEAs + AgNPs 9.86 ± 6.97ab 0.08 ± 0.04b – 19.44 ± 7.09ab – 0.02 ± 0.02b 1.12 ± 0.48b 0.29 ± 0.03b
15%
EEAs + AgNPs 22.81 ± 13.9ab 1.63 ± 0.18b – 14.01 ± 1.6ab – 0.28 ± 0.15b 2.04 ± 0.76ab –
16 Current Trends in Pteridophyte Extracts: From Plant to Nanoparticles
25%
Data shown as mean values ± SE of three replicates
a, b
Interpretation of statistical significance and significant differences through Duncan’s test, p <0.05
349
A. cepa suggested that spore extracts inhibited cytokinesis (Kalcheva et al. 2009).
Formation of binucleate cells was taken into account in genotoxicity analysis, due
to the fact that the disruption of microtubules affects the consolidation of phragmo-
plasts in the telophase (Soliman 2001). Sticky metaphases appeared as a result of
the toxic effect on chromatin and usually led to the death of the cell (Campos et al.
2008). In our study, the frequency of sticky chromosomes was specific to spore
extracts without AgNPs. The variation in the number of chromosomes or chromo-
some sets is a tolerated and relatively common phenomenon in plants, due, at least
partially, to the absence of p53 (Korthout et al. 2002).
The identification of C-mitoses and cells with a chromosome number higher than
2n = 16 in the onion meristematic tips treated with spore extracts containing AgNPs
may have resulted from the interference of some of their compounds with the
assembly and disassembly process of tubulin molecules in the networks of microtu-
bules (Cassimeris et al. 1988). The presence of C-mitoses, along with the variation
in the chromosome number in the experimental samples EEAs + AgNPs, indicates
the antitumour potential of AgNPs.
However, even though higher plants are fundamental test organisms for monitor-
ing and testing genotoxins and the Allium test is marked by sensitivity and repro-
ducibility (Fiskesjö 1985), it is still necessary to evaluate the genotoxic potential of
each compound or bioactive chemical complex, as well as their capacity to damage
the genetic material organized in the form of chromosomes. The evaluation has to
be done through multiple tests that must certify the fidelity of the results (Repetto
et al. 2001).
16.8 Conclusions
Phytosynthesis of Ag nanoparticles in ethanol extracts from spores of Athyrium

filix-femina was demonstrated through SEMChai and EDS analyses. The sizes of
AgNPs varied between 10 and 94 nm. In the meristematic tips of Allium cepa, mito-
sis was inhibited by ethanol extracts from spores. The more pronounced mitode-
pressive effect associated with the presence of C-mitoses was induced by spore
extracts supplemented with AgNPs, which demonstrate potential antitumour effects.
Depending on the absence or the presence of AgNPs, the experimental samples
were defined by distinct chromosomal aberrations, as follows: sticky chromosomes
and binucleate cells and C-mitoses and the variation in the chromosome number,
respectively.
Acknowledgements The authors thank the following researchers for their contribution to the
work: PhD Physicist Cătălin Ducu and PhD Physicist Denis Negrea (University of Pitești). The
spores of Athyrium filix-femina were provided by The Romanian Pteridological Society.
References
Abraham G, Kaushik GK (2015) Antimicrobial activity and identification of potential antimi-

crobial compounds from aquatic pteridophyte, Azolla microphylla Kaulf. Indian J Exp Biol
53:232–235
Aitken RJ, Chaundhry MQ, Boxall ABA, Hull M (2006) Manufacture and use of nanomaterials:
current status in the UK and global trends. Occup Med 56:300–306
Akeroyd J, Synge H (1992) Higher plant diversity. In: Groombridge B (ed) Global biodiversity
status of the Earth's living resources, 1st edn. Springer, Dordrecht, pp 64–87
Alcaraz LE, Blanco SE, Puig ON, Tomas F, Ferretti FH (2000) Antibacterial activity of flavonoids
against methicillin-resistant Staphylococcus aureus strains. J Theor Biol 205:231–240
Alihosseini F, Azarmi S, Ghaffari S, Haghighat S, Sorkhabadi RSM (2016) Synergic antibacterial
effect of curcumin with ampicillin; free drug solutions in comparison with SLN dispersions.
Adv Pharm Bull 6(3):461–465
Allafchian AR, Mirahmadi-Zare SZ, Jalali SHAH, Hashemi SS, Wahabi MR (2016) Green syn-
thesis of silver nanoparticles using phlomis leaf extract and investigation of their antibacterial
activity. J Nanostruct Chem 6(2):129–135
Amoroso VB, Antes DE, Buenavista DP, Coritico FP (2014) Antimicrobial, antipyretic, and anti-
inflammatory activities of selected Philippine medicinal pteridophytes. Asian J Biodivers
5:18–40
Auffan M, Rose J, Wiesner MR, Bottero J (2009) Chemical stability of metallic nanoparticles: a
parameter controlling their potential cellular toxicity in vitro. Environ Pollut 157:1127–1133.
https://doi.org/10.1016/j.envpol.2008.10.002
Bahadori MB, Kordi FM, Ahmadi AA, Bahadori S, Valizadeh H (2015) Antibacterial evalua-
tion and preliminary phytochemical screening of selected ferns from Iran. Res J Pharmacogn
2(2):53–59
Baker C, Pradhan A, Pakstis L, Pochan DJ, Shah SI (2005) Synthesis and antibacterial properties
of silver nanoparticles. J Nanosci Nanotechnol 5(2):244–249
Balaji DS, Basavaraja RD, Mahesh DB, Belawadi KP, Abbaraju V (2008) Biosynthesis and sta-
bilization of Au and Ag alloy nanoparticles by fungus Fusarium semitectum. Sci Technol Adv
Mater 9:035012–035017
Baskaran X, Vigila AVG, Parimelazhagan T, Muralidhara-Rao D, Zhang S (2016) Biosynthesis,
characterization, and evaluation of bioactivities of leaf extact-mediated biocompatible sil-
ver nanoparticles from an early tracheophyte, Pteris tripartita Sw. Int J Nanomedicine
11:5789–5806
Bernhardt ES, Benjamin PC, Hochella MF, Cardinale BJ, Nisbet RM, Richardson CJ, Yin L (2010)
An ecological perspective on nanomaterial impacts in the environment. J Environ Qual 39:1–12
Bhor G, Maskare S, Hinge S, Singh L, Nalawade A (2014) Synthesis of silver nanoparticles by
using leaflet extract of Nephrolepis exaltata L and evaluation of antibacterial activity against
human and plant pathogenic bacteria. Asian J Pharm Technol Innov 2(7), p. 6.
Blajeski AL, Phan VA, Kottke TJ, Kaufmann SH (2002) G1 and G2 cell-cycle arrest following
microtubule depolymerization in human breast cancer cells. J Clin Invest 110(1):91–99. https://
doi.org/10.1172/JCI13275
Braga LC, Leite AA, Xavier KG, Takahashi JA, Bemquerer MP, Chartone-Souza E, Nascimento
AM (2005) Synergic interaction between pomegranate extract and antibiotics against
Staphylococcus aureus. Can J Microbiol 51(7):541–547
Britto JD, Gracelin DHS, Kumar PBJR (2014) Antibacterial activity of silver nanoparticles synthe-
sis from a few medicinal ferns. Bull Environ Pharm Life Sci 3(3):224–227
Bunnag D, Dhorranintra B, Limsuvan S (1989) Ferns and their allergenic importance: skin and
nasal provocation tests to fern spore extract in allergic and non-allergic patients. Ann Allergy
62(6):554–558
Campos JMS, Davide LC, Soares GLG, Viccini LF (2008) Mutagenic effects due to allelopathic
action of fern (Gleicheniaceae) extracts. Allelopath J 22(1):143–151
Cao J, Xia X, Chen X, Xiao J, Wang Q (2013) Characterization of flavonoids from Dryopteris
erythrosora and evaluation of their antioxidant, anticancer and acetylcholinesterase inhibition
activities. Food Chem Toxicol 51:242–250
Cassimeris L, Pryor NK, Salmon ED (1988) Real-time observations of microtubules dynamic
instability in living cells. J Cell Biol 107:2223–2231
Chai TT, Panirchellvum E, Ong H-C, Wong F-C (2012) Phenolic contents and antioxidant proper-
ties of Stenochlaena palustris, an edible medicinal fern. Bot Stud 53:439–446
Chai TT, Yeoh LY, Ismail NIM, Ong HC, Wong FC (2015) Cytotoxicity and antiglucosidase poten-
tial of six selected edible and medicinal ferns. Acta Pol Pharm Drug Res 72(2):297–401
Chang HC, Huang G-J, Agrawal D, Kuo C-L, Wu C-R, Tsay H-S (2007) Antioxidant activities
and polyphenol contents of six folk medicinal ferns used as “Gusuibu”. Bot Stud 48:397–406
Chew FT, Lim SH, Shang HS, Siti Dahlia M, Goh DYT, Lee BW (2000) Evaluation of the allerge-
nicity of tropical pollen and airborne spores in Singapore. Allergy 55:340–347
Chrislyn G, Prachi S, Sangeeta S, Priya S (2016) Green synthesis and characterization of silver
nanoparticles. Int J Adv Res 4(8):1563–1568. https://doi.org/10.21474/IJAR01/1362
Croteau MN, Misra SK, Lioma S, Valsami-Jones E (2014) Bioaccumulation and toxicity of CuO
nanoparticles by a freshwater invertebrate after waterborne and diet borne exposures. Environ
Sci Technol 48:10929–10937. https://doi.org/10.1021/es5018703
Cushnie TPT, Lamb AJ (2005) Antimicrobial activity of flavonoids. Int J Antimicrob Agents
26:343–356
Dalli AK, Saha G, Chakraborty U (2007) Characterization of antimicrobial compounds from a
common fern, Pteris biaurita. Indian J Exp Biol 45(3):285–290
Deliu I, Bejan C, Vișoiu E, Soare LC (2013) The antimicrobial activity of some extracts of fern
gametophytes. Curr Trends Nat Sci 2(2):10–13
Devi S, Yasmeen, Singh J, Shankar R (1989) Patch testing animals to allergenic fern spores.
J Toxicol Cutan Ocul Toxicol 8(2):167–172
Eder M, Mehnert W (1998) Bedeutung planzlicher begleitstoffe in extrackten. Pharmazie
53:285–293
Eneji AE, Inanaga S, Muranaka S, Li J, Hattori T, Tsuji W (2005) Effect of calcium silicate on
growth and dry matter yield of Chloris gayana and Sorghum sudanense under two soil water
regimes. Grass Forage Sci 60:393–398
European Commission Scientific Committee on Emerging and Newly Identified Health Risks
(2006) Modified opinion (after public consultation) on the appropriateness of existing meth-
odologies to assess the potential risks associated with engineered and adventitious products of
nanotechnologies, European Commission, Bruxelles, Belgium, p 3–14
Fernández OL, Mazzeo DEC, Marin-Morales MA (2007) Mechanism of micronuclei formation in
polyploidization cells of Allium cepa exposed to trifluralin herbicide. Pestic Biochem Physiol
88:252–259
Fiskesjö G (1985) The Allium test as standard in environmental monitoring. Hereditas 102:99–112
Fiskesjö G (1993) Allium test I: a 2-3 day plant test for toxicity assessment by measuring the mean
root growth of anions (Allium cepa L.) Environ Toxicol 8(4):461–470. https://doi.org/10.1002/
tox2530080410
Fun S, Tian F, Li J, Hutchins W, Chen H, Yang F, Yuan X, Cui Z, Yang C-H, He C (2017)
Identification of phenolic compounds that suppress the virulence of Xanthomonas oryzae on
rice via the type III secretion system. Mol Plant Pathol 18(4):555–568
Gartner C, Etahl W, Sies H (1997) Lycopene is more bioavailable from tomato paste than from
fresh tomatoes. Am J Clin Nutr 66:116–122
Geethalakshmi R, Sarada DVL (2010) Synthesis of plant mediated silver nanoparticles using
Trianthema decandra extract and evaluation of their anti-microbial activities. Int J Eng Sci
Technol 2(5):970–975
Ghosh S, Patil S, Ahire M et al (2011) Synthesis of gold nanoanisotrops using Dioscorea bulbifera
tuber extract. J Nanomater 2011, Article ID 354793. https://doi.org/10.1155/2011/354793
Ghosh S, Patil S, Ahire M et al (2012a) Synthesis of silver nanoparticles using Dioscorea bul-
bifera tuber extract and evaluation of its synergistic potential in combination with antimicrobial
agents. Int J Nanomedicine 7:483–496
Ghosh S, Patil S, Ahire M et al (2012b) Gnidia glauca flower extract mediated synthesis of gold
nanoparticles and evaluation of its chemocatalytic potential. J Nanobiotechnol 10:17
Glamočlija U, Haverić S, Čakar J, Durmić A, Haverić A, Bajrović K (2014) Bioactivity and geno-
toxicity of centuries old remedy Asplenium scolopendrium L. Int J Pharm 4(2):38–41
Gottschalk F, Sonderer T, Scholz RW, Nowack B (2009) Modeled environmental concentrations
of engineered nanomaterials (TiO, ZnO, Ag, CNT, 2 Fullerenes) for different regions. Environ
Sci Technol 43:9216–9222. https://doi.org/10.1021/es9015553
Hammami S, Snène A, El Mokni R, Faidi K, Falconieri D, Dhaouadi H, Piras A, Mighri Z, Porcedda
S (2016) Essential oil constituents and antioxidant activity of Asplenium ferns. J Chromatogr
Sci 54(8):1341–1345. https://doi.org/10.1093/chromsci/bmw071
Handy RD, Owen R, Valsami-Jones E (2008a) The ecotoxicology of nanoparticles and nanomate-
rials: current satus, knowledge gaps, challenges, and future needs. Ecotoxicology 17:315–325.
https://doi.org/10.1007/s10646-008-0206-0
Handy RD, Kamme F, Lead JR, Hasselov M, Owen R, Crane M (2008b) The ecotoxicology and
chemistry of manufactured nanoparticles. Ecotoxicology 17:287–314. https://doi.org/10.1007/
s10646-008-0199-8
Hattori T, Inanaga S, Araki H, Morita S, Luxuva M, Lux A (2005) Application of silicon enhanced
drought tolerance in Sorghum bicolor. Physiol Plant 123:459–466
Haverkamp R, Marshall A (2009) The mechanism of metal nanoparticle formation in plants: limits
on accumulation. J Nanopart Res 11(6):1453–1463
Ho R, Teai T, Bianchini J-P, Laffont R, Raharivelomanana P (2011) Ferns: from traditional
uses to pharmaceutical development, chemical identification of active principles. In: Kumar
A, Fernández H, Revilla MA (eds) Working with ferns: issues and applications. Springer,
New York, pp 321–346
Höhne H, Richter B (1981) Untersuchungen über den Mineralstoff- und Stickstoffgehalt von
Farnkrautern. Flora 171:1–10
Huang J, Li Q, Sun D, Lu Y, Su Y, Yang X, Wang H, Wang Y, Shao W, Hong NJ, Chen C (2007)
Biosynthesis of silver and gold nanoparticles by novel sundried Cinnamomum camphora leaf.
Nanotechnology 18:105104–105115
IBM Corp Released (2011) IBM SPSS statistics for Windows, version 200 Armonk. IBM Corp,
New York
Jeevanandam J, Chan YS, Danquah MK (2017) Biosynthesis and characterization of MgO nanopar-
ticles from plant extracts via induced molecular nucleation. New J Chem 41(7):2800–2814
Johnson M, Amutha S, Shibila T, Janakiraman N (2017) Green synthesis of silver nanoparticles
using Cyathea nilgirensis Holttum and their cytotoxic and phytotoxic potentials. Part Sci
Technol. https://doi.org/10.1080/02726351.2016.1278292
Jordan MA, Thrower D, Wilson L (1991) Mechanism of inhibition of cell proliferation by Vinca
alkaloids. Cancer Res 51:2212–2222
Kalcheva VP, Dragoeva AP, Kalchev KN, Enchev DD (2009) Cytotoxic and genotoxic effects
of Br-containing oxalosphole on Allium cepa L. root tip cells and mouse bone marrow cells.
Genet Mol Biol 32(2):389–393
Kalita S, Kandimalla R, Sharma KK, Kataki AC, Deka M, Kotoky J (2016) Amoxicillin function-
alized gold nanoparticles reverts MRSA resistance. Mater Sci Eng C 61:720–727
Kandhasamy M, Arunachalam KD, Thatheyus AJ (2008) Drynaria quercifolia (L.) J.Sm: a poten-
tial resource for antibacterial activity. Afr J Microbiol Res 2:202–205
Kang KC, Kim SS, Baik MH, Choi JW, Kwon SH (2008) Synthesis of silver nanoparticles by
using the green chemical method. Appl Chem 12(2):281–284
Khokhani D, Zhang C, Li Y, Wang Q, Zeng Q, Yamazaki A, Hutchins W, Zhou SS, Chen X,

Yang CH (2013) Discovery of plant phenolic compounds that act as type III secretion system
inhibitors or inducers of the fire blight pathogen, Erwinia amylovora. Appl Environ Microbiol
79(18):5424–5436
Klain SJ, Alvarez PJJ, Batley GE, Fernandes TF, Handy RD, Lyon DY, Mahendra S, McLaughlin
MJ, Lead JR (2008) Nanomaterials in the environment: behavior, fate, bioavailability, and
effects. Environ Toxicol Chem 27:1825–1851
Klaine SJ, Koelmans AA, Horne N, Carley S, Handy RD, Kapustka L, Nowack B, Von der
Kammer F (2012) Paradigms to assess the environmental impact of manufactured nanomateri-
als. Environ Toxicol Chem 31(1):3–14. https://doi.org/10.1002/etc.733
Korbekandi H, Chitsazi MR, Asghari G, Najafi RB, Badii A, Iravani S (2014) Green biosynthe-
sis of silver nanoparticles using Azolla pinnata whole plant hydroalcoholic extract. Green
Processes Synth 3(5):365–373. https://doi.org/10.1515/gps-2014-0042
Korthout HA, Casper MP, Kottenhagen MJ, Helmer Q, Wang M (2002) A tormentor in the quest
for plant p-53-like proteins. FEBS Lett 526:53–57
Koteswaramma B, Kamakshamma J, Varalaksmi S (2017) Biological synthesis of silver nanopar-
ticles from aqueous extract of Actiniopteris radiata and evaluation of their antimicrobial activ-
ity. Int J Pharm Biol Sci 8(1):121–125
Krishnaraj C, Ramachandran R, Mohan K, Kalaichelvan PT (2012) Optimization for rapid syn-
thesis of silver nanoparticles and its effect on phytopathogenic fungi. Spectrochim Acta A
93:95–99
Kumar A, Pandey AK, Singh SS, Shanker R, Dhawan A (2011) A flow cytometric method to assess
nanoparticle uptake in bacteria. Cytometry A 79A:707–712
Kunjiappan S, Bhattacharjee C, Chowdhury R (2015) Hepatoprotective and antioxidant effects of
Azolla microphylla based gold nanoparticles against acetaminophen induced toxicity in a fresh
water common carp fish (Cyprinus carpio L.) Nanomed J 2(2):88–110
Lai HY, Lim YY, Tan SP (2009) Antioxidative, tyrosinase inhibiting and antibacterial activities of
leaf extracts from medicinal ferns. Biosci Biotechnol Biochem 73(6):1362–1366
Lai HY, Lim YY, Kim KH (2010) Blechnum orientale Linn a fern with potential as anti-
oxidant, anticancer and antibacterial agent. BMC Complement Altern Med 10:15. http://
wwwbiomedcentralcom/1472-6882/10/15
Lai JC, Lai HY, Rao NK, Ng SF (2016) Treatment for diabetic ulcer wounds using a fern tannin opti-
mized hydrogel formulation with antibacterial and antioxidative properties. J Ethnopharmacol
189:277–289. https://doi.org/10.1016/jep.2016.05.032
Lee HB, Kim JC, Lee SM (2009) Antibacterial activity of two phloroglucinols, flavaspidic
acids AB and PB, from Dryopteris crassirhizoma. Arch Pharm Res 32:655–659. https://doi.
org/10.1007/s12272-009-1502-9
Lee JH, Lim JM, Velmurugan P, Park YJ, Park YJ, Bang KS, Oh BT (2016) Photobiologic-
mediated fabrication of silver nanoparticles with antibacterial activity. J Photochem Photobiol
B 162:93–99. https://doi.org/10.1016/j.jphotobiol.2016.06.029
Lila MA (2009) Interactions between flavonoids that benefit human health. In: Gould K, Davies K,
Winefield C (eds) Anthocyanins biosynthesis, functions, and applications. Springer, New York,
pp 305–324
Lila MA, Raskin I (2005) Health-related interactions of phytochemicals. J Food Sci 7:R20–R27
Lim YH, Kim IH, Seo JJ (2007) In vitro activity of kaempferol isolated from the Impatiens bal-
samina alone and in combination with erythromycin or clindamycin against Propionibacterium
acnes. J Microbiol 45(5):473–477
Liu R (2003) Health benefits of fruit and vegetables are from additive and synergistic combinations
of phytochemicals. Am J Clin Nutr 78:517S–520S
Liu Y, Wujisguleng W, Long C (2012) Food uses of ferns in China: a review. Acta Soc Bot Pol
81(4):263–270
Lok C, Ho C, Chen R, He Q, Tu W, Sun H, Tam PK, Chiu J, Chem C (2007) Silver nanoparticles:
partial oxidation and antibacterial activities. J Biol Inorg Chem 12:527–534
Lu SG (2007) Pteridology. Higher Education Press, Beijing

Ma XY, Xie CX, Liu C, Song JY, Yao H, Luo K, Zhu YJ, Gao T, Pang XH, Qian J, Chen SL (2010)
Species identification of medicinal pteridophytes by a DNA barcode marker, the chloroplast
psbA-trnH intergenic region. Biol Pharm Bull 33(11):1919–1924
Ma H, Williams PL, Diamond SA (2013) Ecotoxicity of manufactured ZnO nanoparticles - a
review. Environ Pollut 172:76–85. https://doi.org/10.1016/j.envpol.2012.08.011
Magaye RR, Wu A, Zhao J, Zou B, Shi H, Yu H, Liu K, Lin X, Xu J, Yang C (2014) Acute toxicity
of nickel nanoparticles in rats after intravenous injection. Int J Nanomedicine 9(1):1393–1402
Makarov VV, Love AJ, Sinitsyna OV, Makarova SS, Yaminsky IV, Taliansky ME, Kalinina NO
(2014) "Green" nanotechnologies: synthesis of metal nanoparticles using plants. Acta Nat
6(1):35–44
Manzo S, Miglietta ML, Rametta G, Buono S, Di Francia G (2013) Toxic effects of ZnO nanopar-
ticles towards marine algae Dunaliella tertiolecta. Sci Total Environ 445-446:371–376. https://
doi.org/10.1016/j.scitotenv.2012.12.051
Marston A, Hostettmann K (2006) Separation and quantification of flavonoids. In: Andersen ØM,
Markham KR (eds) Flavonoids chemistry, biochemistry and applications. Taylor & Francis
Group, Boca Raton, London, New York, pp 1–36
McFee AF, Tice RR (1990) Influence of treatment to sacrifice time and the presence of BrdUrd on
chemically-induced aberration rates in mouse marrow cells. Mutat Res 241:95–108
Mittal AK, Chisti Y, Banerjee UC (2013) Synthesis of metallic nanoparticles using plant extracts.
Biotechnol Adv 31:346–356. https://doi.org/10.1016/j.biotechadv.2013.01.003
Moran RC (2004) A natural history of ferns Portland. Timber Press, Portland
Moran RC (2008) Diversity, biogeography, and floristics. In: Ranker TA, Haufler CH (eds)
Biology and evolution of ferns and Lycophytes. Cambridge University Press, New York,
pp 367–394
Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Ramirez JT, Yacaman MJ (2005)
The bactericidal effect of silver nanoparticles. Nanotechnology 16(10):2346–2353. https://doi.
org/10.1088/0957-4484/16/10/059
Nalwade AR, Badhe MN, Pawale CB, Hinge SB (2013) Rapid biosynthesis of silver nanopar-
ticles using fern leaflet extract and evaluation of their antibacterial activity. Int J Biol Technol
4(2):12–18
Nath K, Bhattacharya MK, Sen A, Kar S (2013) Antibacterial activity of frond extract of Asplenium
nidus L., a threatened ethnomedicinal fern of North East India. International. J Pharmacogn
Phytochem 28(2):1169–1172
Nayak N, Rath S, Mishra MP, Ghosh G, Padhy RN (2013) Antibacterial activity of the terrestrial
fern Lygodium flexuosum (L.) Sw. against multidrug resistant enteric- and uro- pathogenic
bacteria. J Acute Dis 2:270–276
Nowack B, Bucheli TD (2007) Occurrence, behavior and effect of nanoparticles in the environ-
ment. Environ Pollut 150(1):5–22
Pal SK (2012) Study of solvent extracts of some selected ferns for antimicrobial activity. Indian
J Biol Sci 18:33–37
Pan C, Chen YG, Ma XY, Jiang JH, He F, Zhang Y (2011) Phytochemical constituents and
pharmacological activities of plants from the genus Adiantum: a review. Trop J Pharm Res
10(5):681–692
Panneerselvam C, Murugan K, Roni M, Aziz AT, Suresh U, Rajaganesh R, Mathiyazhagan P,
Subramaniam J, Devakumar D, Nicoletti M, Higuchi A, Alarfaj AA, Munusamy MA, Kumar S,
Desneux N, Benelli G (2016) Fern-synthesized nanoparticles in the fight against malaria: LC/
MS analysis of Pteridium aquilinum leaf extract and biosynthesis of silver nanoparticles with
high mosquitocidal and antiplasmodial activity. J Parasitol Res 115(3):997–1013. https://doi.
org/10.1007/s00436-015-4828-x
Parihar P, Parihar L, Bohra A (2006) Antibacterial activity of Athyrium pectinatum (Wall) Presl.
Nat Prod Rad 5(4):262–265
Park Y, Hong YN, Weyers A, Kim YS, Linhardt RJ (2011) Polysaccharides and phytochemicals: a
natural reservoir for the green synthesis of gold and silver nanoparticles. IET Nanobiotechnol
5(3):69–78. https://doi.org/10.1049/iet-nbt.2010.0033
Prathna TC, Mathew L, Chandrasekaran N, Raichur AM, Mukherjee A (2010)
Biomimetic synthesis of nanoparticles: science, technology and applicabil-
ity. In: Mukherjee A (ed) Biomimetics learning from nature. InTech. https://doi.
org/10.5772/8776. https://www.intechopen.com/books/biomimetics-learning-from-nature/
biomimetic-synthesis-of-nanoparticles-science-technology-amp-applicability
Rajaganesh R, Murugan K, Panneerselvam C, Jayashanthini S, Aziz AT, Roni M, Suresh U, Trivedi
S, Rehman H, Nicoletti M, Benelli G, Higuchi A (2016) Fern-synthesized silver nanocrystals:
towards a new class of mosquito oviposition deterrents? Res Vet Sci 109:40–51. https://doi.
org/10.1016/j.rvsc.2016.09.012
Rajesh KD, Vasantha S, Panneerselvam A, Rajesh NV, Jeyathilakan N (2016) Phytochemical
analysis, in vitro antioxidant potential and gas chromatography-mas spectrometry studies of
Dicranopteris linearis. Asian J Pharm Clin Res 9(Suppl. 2):1–6
Rani D, Khare PB, Dantu PK (2010) In vitro antibacterial and antifungal properties of aqueous and
non-aqueous frond extracts of Psilotum nudum, Nephrolepis biserrata and Nephrolepis cordi-
folia. Indian J Pharm Sci 72(6):818–822. https://doi.org/10.4103/0250-474X.84606
Rank J (2003) The method of Allium anaphase-telophase chromosome aberration assay. Ekologija
(Vilnius) 1:38–42
Raut R, Kolekar N, Lakkakula J, Mendhulkar V, Kashid S (2010) Extracellular synthesis of silver
nanoparticles using dried leaves of Pongamia pinnata (L.) Pierre. Nano-Micro Lett 2(2):106–
113. https://doi.org/10.5101/nml.v2i2.p106-113
Repetto G, Jos A, Hayen MJ, Molero ML, del Peso A, Salguero M, del Castillo P, Rodriguez-
Vicente MC, Repetto M (2001) A test battery for the ecotoxicological evaluation of pentachlo-
rophenol. Toxicol In Vitro 15:503–509. https://doi.org/10.1016/S0887-2333(01)00055-8
Roco MC (2005) Environmentally responsible development of nanotechnology. Environ Sci
Technol 39(5):106A–112A. https://doi.org/10.1021/es053199u
Roos M (1996) Mapping the world’s pteridophyte diversity - systematics and floras. In: Camus JM,
Gibby M, Johns RJ (eds) Pteridology in perspective. Kew: Royal Botanic Gardens, London,
pp 29–42
Roudsari MT, Bahrami AR, Dehghani H, Iranshani M, Matin MM, Mahmoudi M (2012) Bracken-
fern extract induce cell cycle arrest and apoptosis in certain cancer cell lines. Asian Pac
J Cancer Prev 13(12):6047–6053
Samidoss CM, Murugn K, Mathath R, Sivaprizajothi S, Suganya NA (2013) Larvicidal potential of
silver nanoparticles synthesized using Adiantum capillus-veneris against Anopheles stephensi
(Diptera; Culicidae). Int J Curr Trop Med Health Res 1(1):9–18
Sant DG, Gujarathi TR, Harne SR, Ghosh S, Kitture R, Kale S, Chopade BA, Pardesi KR (2013)
Adiantum philippense L. frond assisted rapid green synthesis of gold and silver nanoparticles.
J Nanopart 2013:9. https://doi.org/10.1155/2013/182320
Santhoshkumar S, Nagarajan N (2014) AM fungal association in the rhizosphere soil of some
pteridophytic plant species in Valparai Hills, Western Ghats of Tamilnadu, India. Int J Life Sci
2(3):201–206
Santos ATB, Silva Araujo TF, Silva LCN, Silva CB, Oliveira AFM, Araujo JM, Menezes Lima VL,
Santos Correia MT (2015) Organic extract from Indigofera suffruticosa leaves have antimicro-
bial and synergic actions with erythromycin against Staphylococcus aureus. Front Microbiol
6:1–7. https://doi.org/10.3389/fmicb.2015.00013
Santos RD, Pimenta-Freire G, FJB F, Dias-Souza MV (2016) Interference of flavonoids and carot-
enoids on the antimicrobial activity of some drugs against clinical isolates of Pseudomonas
aeruginosa. Int Food Res J 23(3):1268–1273
Schneider H, Schuettpelz E, Pryer KM, Cranfill R, Magallón S, Lupia R (2004) Ferns diversified in
the shadow of angiosperms. Nature 428(6982):553–557. https://doi.org/10.1038/nature02361
Simán SE, Povey AC, Ward TH, Margison GP, Sheffield E (2000) Fern spore extracts can damage
DNA. Br J Cancer 83(1):69–73
Sinha T, Ahmaruzzaman M (2016) Indigenous north eastern Indian fern mediated fabrication
of spherical silver and anisotropic gold nano structured materials and their efficiency for the
abatement of perilous organic compounds from waste eater – a green approach. RSC Adv
6:21076–21089. https://doi.org/10.1039/C5RA26124D
Soare LC, Ferdeș M, Deliu I, Gibea A (2012a) Studies regarding the antibacterial activity of some
extracts of native pteridophytes. UPB Sci Bull Ser B 74(1):21–26
Soare LC, Ferdeș M, Stefanov S, Denkova Z, Nikolova R, Denev P, Bejan C, Păunescu A (2012b)
Antioxidant activity, polyphenols content and antimicrobial activity of several native pterido-
phytes of Romania. Not Bot Hort Agrobot Cluj Napoca 40(1):53–57. https://doi.org/10.15835/
nbha4016648
Soare LC, Deliu I, Iosub I, Dobrescu CM, Ferdeș M (2012c) New therapeutic formulations with an
antibacterial effect, based on plant extracts. Current trends in. Nat Sci 1(2):79–82
Soliman MI (2001) Genotoxicity testing of neem plant (Azadirachta indica A. Juss) using the
Allium cepa chromosome aberration assay. J Biol Sci 1(11):1021–1027
Souri E, Amin G, Farsam H, Jalalizadeh H, Barezi S (2008) Screening of thirteen medicinal plant
extracts for antioxidant activity. Iran J Pharm Res 7(2):149–154
Șuțan AN, Fierăscu I, Fierăscu RC, Manolescu DȘ, Soare LC (2016) Comparative analytical
characterization and in vitrocytogenotoxic activity evaluation of Asplenium scolopendrium L.
leaves and rhizome extracts prior to and after Ag nanoparticles phytosynthesis. Ind Crop Prod
83:379–386. https://doi.org/10.1016/j.indcrop.2016.01.011
Tedesco SB, Laughinghouse HD IV (2012) Bioindicator of genotoxicity: the Allium cepa test. In:
Srivastava JK (ed) Environmental contamination. InTech, Rijeka, pp 137–156
Teow SY, Ali SA (2015) Synergistic antibacterial activity of Curcumin with antibiotics against
Staphylococcus aureus. Pak J Pharm Sci 28(6):2109–2114
Thakar SB, Ghorpade PN, Kale MV, Sonawane KD (2015) Fern Ethnomedicinal Plant Database:
exploring fern ethnomedicinal plants knowledge for computational drug discovery. Curr
Comput Aided Drug Des 11(3):266–271
Tryon AF, Lugardon B (1978) Wall structure and mineral content in Selaginella spores. Pollen
Spores 20:315–340
Turkevich J, Stevenson PC, Hiller PCJ (1951) A study of the nucleation and growth processes in
the synthesis of colloidal gold. Discuss Faraday Soc 11:55
Valizadeh H, Sonboli A, Kordi FM, Dehghan H, Bahadori MB (2015) Cytotoxicity, antioxidant
activity and phenolic content of eight fern species from North of Iran. Pharm Sci 21:18–24.
https://doi.org/10.15171/PS.2015.12
Verma DK, Hasan SH, Banik RM (2016) Photo-catalyzed and phyto-mediated rapid green syn-
thesis of silver nanoparticles using herbal extract of Salvinia molesta and its antimicrobial effi-
cacy. J Photochem Photobiol B 155:51–59. https://doi.org/10.1016/j.jphotobiol.2015.12.008
Wang S, Lawson R, Ray P, Yu H (2011) Toxic effects of gold nanoparticles on Salmonella
typhimurium bacteria. Toxicol Ind Health 27(6):547–554. https://doi.org/10.1177/074823371
0393395
Xavier GSA, Selvaraj P, Nida J (2016) Impact of phytoecdysone fractions of the ferns Cyclosorous
interruptus, Christella dentata and Nephrolepis cordifolia on the biology of Spodoptera litura
(Fab). J Biopest 9(2):125–134
Xie Y, Zheng Y, Dai X, Wang Q, Cao J, Xiao J (2015) Sesonal dynamics of total flavonoid contents
and antioxidant activity of Dryopteris erythrosora. Food Chem 186:113–118
Yoshida T, Hatano T, Ito H (2005) High molecular weight plant polyphenols (tannins): prospec-
tive functions. In: Romeo JT (ed) Recent advances in phytochemistry, volume 39: chemical
ecology and phytochemistry of forest ecosystems. Elsevier Inc., San Diego, CA, pp 163–190.
Yusuf UK (1994) Flavonoid glycosides in the fern Blechnum orientale Linn. Am Fern J 84:69–70

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Chapter 16

Current Trends in Pteridophyte Extracts:

The diversity of pteridophytes is estimated at 10,000–15,000 species (Akeroyd and

L. C. Soare (*) · N. A. Șuțan

© Springer International Publishing AG, part of Springer Nature 2018 329

16.2 Crude Extracts

Extracts were obtained, and phytochemical characterization along with bioactiv-

Pteridophytes contain a wide range of bioactive compounds, from the terpenoid,

important antimicrobial activity against multidrug-resistant bacteria such as

between curcumin and some synthetic antibiotics produced synergetic antibacterial

The pH value of the reaction mixture has an extraordinary influence on the

Up to the present, numerous studies have promoted fern extracts as renewable

Several studies have evaluated the mosquitocidal effect of nanoparticles whose

Average root lengths (mm) 25.00

flexuosa and Gleicheniella pectinata through their allelopathic action on meriste-

extracts supplemented with AgNPs through biosynthesis induced the accumulation

Phytosynthesis of Ag nanoparticles in ethanol extracts from spores of Athyrium

Abraham G, Kaushik GK (2015) Antimicrobial activity and identification of potential antimi-

Khokhani D, Zhang C, Li Y, Wang Q, Zeng Q, Yamazaki A, Hutchins W, Zhou SS, Chen X,

Lu SG (2007) Pteridology. Higher Education Press, Beijing

You might also like

Citação Artigo 2 Flavonoides e Carotenoides IFRJ - 2 PDF

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

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Is this content inappropriate?

Copyright:

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Citação Artigo 2 Flavonoides e Carotenoides IFRJ - 2 PDF

Uploaded by

Copyright:

Available Formats

Chapter 16

Current Trends in Pteridophyte Extracts:

The diversity of pteridophytes is estimated at 10,000–15,000 species (Akeroyd and

L. C. Soare (*) · N. A. Șuțan

© Springer International Publishing AG, part of Springer Nature 2018 329

16.2 Crude Extracts

Extracts were obtained, and phytochemical characterization along with bioactiv-

Pteridophytes contain a wide range of bioactive compounds, from the terpenoid,

i­mportant antimicrobial activity against multidrug-resistant bacteria such as

between curcumin and some synthetic antibiotics produced synergetic antibacterial

The pH value of the reaction mixture has an extraordinary influence on the

Up to the present, numerous studies have promoted fern extracts as renewable

Several studies have evaluated the mosquitocidal effect of nanoparticles whose

Average root lengths (mm) 25.00

flexuosa and Gleicheniella pectinata through their allelopathic action on meriste-

extracts supplemented with AgNPs through biosynthesis induced the accumulation

Phytosynthesis of Ag nanoparticles in ethanol extracts from spores of Athyrium

Abraham G, Kaushik GK (2015) Antimicrobial activity and identification of potential antimi-

Khokhani D, Zhang C, Li Y, Wang Q, Zeng Q, Yamazaki A, Hutchins W, Zhou SS, Chen X,

Lu SG (2007) Pteridology. Higher Education Press, Beijing

You might also like

16.2 Crude Extracts

important antimicrobial activity against multidrug-resistant bacteria such as